The interactions between pathogens and hosts lead to a massive upregulation of antimicrobial host effector molecules. Among these, the 65 kDa guanylate binding proteins (GBPs) are interesting candidates as intricate components of the host effector molecule repertoire. Members of the GBP family are highly conserved in vertebrates. Previous reports indicate an antiviral activity of human GBP1 (hGBP1) and murine GBP2 (mGBP2). We recently demonstrated that distinct murine GBP (mGBP) family members are highly upregulated upon Toxoplasma gondii infection and localize around the intracellular protozoa T. gondii. Moreover, we characterised five new mGBP family members within the murine 65 kDa GBP family. Here, we identified a new mGBP locus named mGbp11. Based on bacterial artificial chromosome (BAC), expressed sequence tag (EST), and RT-PCR analyses this study provides a detailed insight into the genomic localization and organization of the mGBPs. These analyses revealed a 166-kb spanning region on chromosome 3 harboring five transcribed mGBPs (mGbp1, mGbp2, mGbp3, mGbp5, and mGbp7) and one pseudogene (pseudomGbp1), as well as a 332-kb spanning region on chromosome 5 consisting of six transcribed mGBPs (mGbp4, mGbp6, mGbp8, mGbp9, mGbp10, and mGbp11), and one pseudogene (pseudomgbp2). Besides the strikingly high homology of 65% to 98% within the coding sequences, the mGBPs on chromosome 5 cluster also exhibit a highly homologous exon-intron structure whereas the mGBP on chromosome 3 reveals a more divergent exon-intron structure. This study details the comprehensive genomic organization of mGBPs and suggests that a continuously changing microbial environment has exerted evolutionary pressure on this gene family leading to multiple gene amplifications. A list of links for this article can be found in the Availability and requirements section.